Entinostat as a combinatorial therapeutic for rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. For the alveolar subtype (ARMS), the presence of the PAX3::FOXO1 fusion gene and/or metastases are strong predictors of poor outcome. Metastatic PAX3::FOXO1+ ARMS often responds to chemotherapies initially, only to subsequently relapse and become resistant with most patients failing to survive beyond 8 years post-diagnosis. No curative intent phase II or phase III clinical trial has been available for patients in the past 10 years (ARST0921). Thus, metastatic ARMS represents a significantly unmet clinical need. Chemotherapy resistance in ARMS has previously been attributed to PAX3::FOXO1-mediated cell cycle checkpoint adaptation, which is mediated by an HDAC3-SMARCA4-miR-27a-PAX3::FOXO1 circuit that can be disrupted by HDAC3 inhibition. In this study, we investigated the therapeutic efficacy of combining the epigenetic regulator entinostat, a Class I Histone Deacetylase (HDAC1-3) inhibitor, with RMS-specific chemotherapies in patient derived xenograft (PDX) models of RMS. We identified single agent, additive or synergistic relationships between relapse-specific chemotherapies and clinically relevant drug exposures of entinostat in three PAX3::FOXO1+ ARMS mouse models. This preclinical data provides further rationale for clinical investigation of entinostat, already known to be well tolerated in a pediatric phase I clinical trial (ADVL1513).

SMARCA4, which in turn inhibits miR-27a, resulting in sustained expression of the PAX3::FOXO1 fusion oncogene.Inhibition of this pathway using entinostat decreases SMARCA4 and allows for the expression of miR-27a, resulting in chemotherapy sensitization in a time frame of 72 h (Fig. 1) 3 and as a separate process from longerterm chromatin remodeling [11][12][13][14] .Separately, Bharathy and colleagues also observed single agent activity of entinostat for 50% of fusion negative RMS models, keeping with related reports of HDAC3 inhibition in ERMS 15,16 .
We sought to expand on these studies by newly investigating the therapeutic efficacy of entinostat in combination with a series of rhabdomyosarcoma relapse-related chemotherapies in both ARMS and ERMS in vivo which could support the development of a human subject clinical trial in refractory and relapsed rhabdomyosarcoma.In pediatric phase I clinical trials (ADVL1513) the maximum tolerated dose (MTD) of entinostat for children was greater than for adults with no dose-limiting toxicity 17 .The preclinical study here demonstrates single agent activity or activity of entinostat in combination with current standard of care chemotherapeutics.

Results
The entinostat dosage approximating human AUC with a non-zero trough is 4 mg/kg po daily The illustrative framework for drug dosing, PDX models and compounds tested is presented in Fig. 2A-E.In humans, pediatric patients given the recommended phase II dose (RP2D) of entinostat of 4 mg/m 2 orally (po) administered once weekly have a C max of 53 ng/mL (140.8 nM) with a t 1/2 of 45 h, and a corresponding drug exposure (area under the curve, AUC) of 1162 ng*h/mL (3087 nM*h) (Fig. 2A) 17 .In mice, the plasma concentration-time profile following administration of a single dose or multiple doses of entinostat by oral gavage at 4 mg/kg is presented in Figs.2B and 3.The C max of 4710 ± 1260 nM occurred at 0.25 h.The AUC 0-12 h and AUC 0-24 h from single dose was 2470 nM*h and 2510 nM*h, respectively.The trough concentrations at 24, 72, and 96 h were 0.45 ± 0.05 nM, 1.46 ± 0.47 nM and 2.79 ± 0.66 nM, respectively.When correcting mouse exposure based on the value of unbound fraction of 0.38 18 , the C max for unbound entinostat was 1576 ± 476 nM, and the AUC 0-12 h and AUC 0-24 h were 934 nM*h and 949 nM*h, respectively.Trough concentrations at 24, 72, and 96 h were 0.17 ± 0.02 nM, 0.56 ± 0.18 nM and 1.06 ± 0.25 nM, respectively.In previous studies, a trough level > 1 nM was pivotal to maintaining PAX3::FOXO1 protein level suppression 3 .Therefore, prioritizing a murine AUC which is at or below the pediatric AUC and a ≥ 1 nM threshold trough level to model entinostat dosing between humans and mice, the oral gavage of 4 mg/kg was selected for combination studies in mice.

The patient-derived xenograft models used in this study reflect stage IV and recurrent disease
Patient derived xenograft models were selected for implantation (Table 1).CTG-1008, CTG-1916, and CTG-2127 were selected as ARMS models, all featuring the PAX3::FOXO1 fusion.No PAX7::FOXO1 lines were available for implantation at the time of this study.CTG-1008 (alias CF-00071) was a recurrent stage IV tumor of the uterus biopsied from a 11-year-old female.CTG-1916 (alias CF-01513) was a recurrent stage IV tumor of the arm biopsied from a 5-year-old female.CTG-2127 was a stage IV tumor of the groin biopsied from a 12-year-old female.For comparison, CTG-1213 (alias CF-00072) was established from a biopsy of a 2-year-old female with stage IV ERMS of the vagina.CTG-1213 harbors an NRAS Q61K mutation.CTG-1213 served as a control for the effect of HDAC inhibition on a fusion negative tumor.

PDX models of ARMS respond to entinostat as a single agent or in combination with chemotherapies
Single agent entinostat and entinostat-chemotherapy efficacy in tumor-bearing PDX models of ARMS are given Fig. 4 (CTG-1008), Fig. 5 (CTG-1916) and Fig. 6 (CTG-2127).The impact of treatment on body weight during the course of study is provided in Figures S1-S3.The entinostat dose and interval were guided by results from Fig. 3, which represents the plasma concentration-time profile following administration of a single dose of entinostat of 4 mg/kg in mice, whereas chemotherapy doses of vinorelbine, cyclophosphamide, doxorubicin and topotecan were selected intentionally to give an intermediate (non-curative) response so that the effect of entinostat could be observed and statistically tested.In general, all monotherapy and combination therapy regimens were well-tolerated.In Fig. 4, 5 and 6, one-way comparison p-values (single factor ANOVA analysis) are given for key comparisons of efficacy.Tables of complete statistical comparisons of efficacy and body weight monitoring for all PDX testing are given in Supplemental Tables 1-6.
For evaluation of entinostat monotherapy, models CTG-1008 (Fig. 4F) and CTG-1916 (Fig. 5F) had no single agent activity in line with previously-published studies showing that PAX3::FOXO1 is dispensable for tumor maintenance in the short term 4,19,20 .Unexpectedly, ARMS model CTG-2127 was exquisitely sensitive to entinostat monotherapy (Fig. 6F; p = 0.053 unadjusted).For combination therapies, the exquisite sensitivity of CTG-2127 to monotherapy made evaluations of drug synergy not possible.However, for model CTG-1008, the combination of entinostat with vinorelbine and the combination of entinostat with cyclophosphamide reached statistical significance before adjusting for multiple comparisons (Fig. 4G, H; p = 0.016 and 0.028 unadjusted, respectively).Possible trends existed for the combination of entinostat with doxorubicin and the combination of entinostat with topotecan (Fig. 4I, J; p = 0.138 and 0.236 unadjusted, respectively).For model CTG-1916, a trend was present for the combination of entinostat with cyclophosphamide (Fig. 5H; p = 0.136 unadjusted).

A single PDX model of ERMS responded modestly to entinostat as a single agent and in combination with chemotherapies
Single agent entinostat and entinostat-chemotherapy efficacy in a single tumor-bearing PDX model of ERMS is given Fig. 7 (CTG-1213).The impact of treatment on body weight during the course of study is provided in Fig. S4.As was used in ARMS PDX testing, the entinostat dose and interval were guided by results from Fig. 3, and chemotherapy doses were selected intentionally to give an intermediate response so that the effect of entinostat could be observed.As was used in ARMS PDX testing, monotherapy and combination therapy regimens were generally well-tolerated.In Fig. 7, one-way comparison p-values (single factor ANOVA analysis) are given for key comparisons of efficacy.Tables of complete statistical comparisons of efficacy and body weight monitoring for all PDX testing are given in Supplemental Tables 7, 8.
For evaluation of entinostat monotherapy, model CTG-1213 had a statistically significant response (Fig. 7F; p = 0.019 unadjusted).In previously published PDX studies, an entinostat monotherapy response was observed in 2 of 4 fusion negative RMS models 15 .For combination therapies, a trend was observed for the combination of entinostat with cyclophosphamide versus cyclophosphamide alone (Fig. 7H; p = 0.109 unadjusted) and the combination of entinostat with vinorelbine (Fig. 7G; p = 0.291 unadjusted).

Entinostat and mocetinostat are mechanistically comparable with respect to reducing PAX3::FOXO1
Because the similar Class I/IV HDAC inhibitor, mocetinostat, has shown activity in combination with vinorelbine in an ongoing phase I clinical trial of patients with refractory or recurrent RMS 21 , we tested whether mocetinostat had similar mechanistic activity to decrease PAX3::FOXO1 protein.For the ARMS cell line Rh30 treated for 72 h, both entinostat and mocetinostat reduced PAX3::FOXO1 protein in a comparable, dose-dependent manner (Fig. S5).
The study presented here investigates the role of entinostat, a Class I/III HDAC inhibitor, as a single agent or in combination with 4 other chemotherapeutic drugs in 3 ARMS and 1 ERMS PDX models.Entinostat demonstrated efficacy when used as a single agent in one of the ARMS PDX models and in combination with other chemotherapeutics (vinorelbine, cyclophosphamide, doxorubicin and topotecan) for 2 of the 3 ARMS PDX models tested that were not already exquisitely sensitive to entinostat as a monotherapy.In the ERMS PDX model, entinostat exhibited a modest effect.Pharmacokinetic modeling between humans and mice is often a challenge, which we addressed here with a clear rationale for the murine dose selected: we have compared the ADVL1513 pediatric phase I RP2D of 4 mg/m 2 po weekly entinostat 17 to the pharmacokinetics in the mouse at a dose of 4 mg/kg po daily, finding a shorter half-life in mice, a higher C max in mice, a somewhat lower AUC in mice, yet a similar non-zero trough that supports the mechanism of entinostat to suppress PAX3::FOXO1 protein levels.The 4 mg/kg po daily dose is the same as we have published in previous preclinical studies for which 7 of 7 contemporary patientderived xenografts (mostly autopsy-derived) showed additive or synergistic activity of entinostat in combination with the chemotherapy vincristine in vivo for ARMS 3 .For ERMS, single agent activity was observed for 2 of 4 contemporary patient-derived xenografts (mostly relapse-derived) in a previously reported study 15 .In contrast, another group's study 23 used a different dosing regimen and had notable design limitation: First, the pharmacodynamic studies were done with two alveolar rhabdomyosarcoma PDX models (Rh10 and Rh65) that lacked baseline PAX3::FOXO1 protein expression in 1 of 3 control animals for each model-drawing concerns for the authenticity of these models.In efficacy studies, only two ARMS PDX models (Rh10 and Rh41) were used, despite these models not being contemporary.Most importantly, for dosing (despite the known shorter murine half-life of entinostat) mice were given 3-day drug holidays per week and mouse dosing was only given 4 days per week at 2.5 mg/kg po BID.The lack of efficacy of entinostat plus vincristine in these mouse models was therefore not unexpected.
In our current study, entinostat monotherapy had no effect on tumor growth in 2 ARMS models, which is consistent with prior published observations that PAX3::FOXO1 is to a degree dispensable for tumor maintenance until PAX3::FOXO1 expression goes to absolute zero (i.e., as seen comparing efficient RNA interference studies vs CRISPR) 4,19,20 (depmap.org).However, in one ARMS model entinostat monotherapy had unexpected, nearcomplete efficacy to suppress tumor growth.For ERMS, one tested ERMS PDX model showed modest growth inhibition, which is in keeping with the ~ 50% response rate of fusion negative RMS that has been published 15 .
To test whether the synergies seen between entinostat, and vincristine extend to other rhabdomyosarcomaspecific, relapse-oriented chemotherapies, we tested entinostat with vinorelbine, cyclophosphamide, doxorubicin and topotecan in 3 ARMS PDX models and 1 ERMS PDX model.For ARMS, the exquisite sensitivity of one ARMS PDX model to entinostat abrogated evaluation of entinostat-mediated chemotherapy sensitization; however, in the 2 evaluable ARMS PDX models, a significant difference or trend was observed for the combination being greater than the efficacy of the single chemotherapy tested for each of the chemotherapies.
From a clinical trial concept perspective, correlative studies with ongoing rhabdomyosarcoma clinical trials are informative: the class I/IV HDAC inhibitor mocetinostat, has shown activity in combination with vinorelbine in an ongoing phase I clinical trial of patients with refractory or recurrent RMS 21 .For this study, enrollment on the dose escalation cohort has been completed and enrollment is ongoing for the dose expansion cohort.To date, 8 patients have been enrolled and 7 have had response evaluation.Of these 7 patients, 4 have had partial response (PR) and 2 have had stable disease (SD) for a disease control rate (DCR) of 86% with median duration of control of 8 months.To date, the only grade 3 or 4 treatment related adverse effects have been neutropenia, anemia and nausea.Neutropenia was transient and responsive to growth factors 21 .In our laboratory studies, mocetinostat and entinostat were comparable in the mechanistic effect of suppressing PAX3::FOXO1 levels.At present, mocetinostat is no longer accessible for clinical trials or commercial development, thereby restricting further examination in this population.Consequently, only entinostat was utilized in the murine PDX models.
On whole, these preclinical studies had the common challenge of matching human and murine pharmacokinetics, as well as the limitation of using only 3 ARMS PDX models and 1 ERMS PDX model.However, these studies are in keeping with extensive other PDX studies 3,15 , support a general observation that entinostat complements a range of chemotherapeutics, and show the mechanistic equivalency of entinostat and mocetinostat, another HDAC inhibitor with observed clinical efficacy in RMS.Taken together, these combined results support the ongoing development of entinostat for curative-intent clinical trials of entinostat plus chemotherapeutics for RMS.

Pharmacokinetics
Mice were dosed with entinostat at 4 mg/kg or vehicle control via oral gavage once a day.Mice (n = 3/time point) were euthanized at 0.25, 0.5, 1, 2, 4, 8, 12, 16, 20 and 24 h after a single dose.Additional samples were obtained prior to the daily dose on Day 4 (72 h) and 5 (96 h).Entinostat was extracted from plasma (using sodium heparin) and analyzed by LC/MS/MS as previously described over the range of 0.3-266 nM with dilutions of 1:100

Figure 2 .
Figure 2. (A) Illustrative representation of entinostat pharmacokinetics in humans derived from clinical trial and preclinical data 17 .(B) Illustrative representation of entinostat pharmacokinetics in mice derived from clinical and preclinical data.(C) Patient derived xenograft mouse models of rhabdomyosarcoma (CTG-1008, CTG-1916, CTG-2127 and CTG1213).(D) Chemical structure of entinostat.(E) Chemical structure of mocetinostat.(illustration by Sun Young Park).

Figure 3 .
Figure 3. Concentration-time profiles of entinostat in mice (n = 3) treated with multiple doses of 4 mg/kg administered orally once daily.Plasma was obtained at timepoints over 24 h after a single dose as well as prior to the daily dose on Day 4 (72 h) and Day 5 (96 h).Data points and error bars represent mean and SD of 3 replicates, respectively.